Repurposing SGLT2 inhibitors for autoimmune diseases? YES, WE MAY!

T cells play a key role in driving autoimmunity, with alterations in metabolism powering their effector function. In the July 11 issue of Cell Metabolism, Jenkins et al.1 describe how a type 2 diabetes drug, canagliflozin, can be repurposed for the treatment of autoimmune disorders through metabolic reprogramming of the T cell response.

Obesity defined molecular endotypes in the synovium of patients with osteoarthritis provides a rationale for therapeutic targeting of fibroblast subsets.

BACKGROUND: Osteoarthritis (OA), a multifaceted condition, poses a significant challenge for the successful clinical development of therapeutics due to heterogeneity. However, classifying molecular endotypes of OA pathogenesis could provide invaluable phenotype-directed routes for stratifying subgroups of patients for targeted therapeutics, leading to greater chances of success in trials. This study establishes endotypes in OA soft joint tissue driven by obesity in both load-bearing and non-load bearing joints. METHODS: Hand, hip, knee and foot joint synovial tissue was obtained from OA patients (n = 32) classified as obese (BMI > 30) or normal weight (BMI 18.5-24.9). Isolated fibroblasts (OA SF) were assayed by Olink proteomic panel, seahorse metabolic flux assay, Illumina's NextSeq 500 bulk and Chromium 10X single cell RNA-sequencing, validated by Luminex and immunofluorescence. RESULTS: Targeted proteomic, metabolic and transcriptomic analysis found the inflammatory landscape of OA SFs are independently impacted by obesity, joint loading and anatomical site with significant heterogeneity between obese and normal weight patients, confirmed by bulk RNAseq. Further investigation by single cell RNAseq identified four functional molecular endotypes including obesity specific subsets defined by an inflammatory endotype related to immune cell regulation, fibroblast activation and inflammatory signaling, with up-regulated CXCL12, CFD and CHI3L1 expression. Luminex confirmed elevated chitase3-like-1(229.5 vs. 49.5 ng/ml, p < .05) and inhibin (20.6 vs. 63.8 pg/ml, p < .05) in obese and normal weight OA SFs, respectively. Lastly, we find SF subsets in obese patients spatially localise in sublining and lining layers of OA synovium and can be distinguished by differential expression of the transcriptional regulators MYC and FOS. CONCLUSION: These findings demonstrate the significance of obesity in changing the inflammatory landscape of synovial fibroblasts in both load bearing and non-load bearing joints. Describing multiple heterogeneous OA SF populations characterised by specific molecular endotypes, which drive heterogeneity in OA disease pathogenesis. These molecular endotypes may provide a route for the stratification of patients in clinical trials, providing a rational for the therapeutic targeting of specific SF subsets in specific patient populations with arthritic conditions.

Understanding lactate sensing and signalling.

Metabolites generated from cellular and tissue metabolism have been rediscovered in recent years as signalling molecules. They may act as cofactor of enzymes or be linked to proteins as post-translational modifiers. They also act as ligands for specific receptors, highlighting that their neglected functions have, in fact, a long standing in evolution. Lactate is one such metabolite that has been considered for long time a waste product of metabolism devoid of any biological function. However, in the past 10 years, lactate has gained much attention in several physio-pathological processes. Mechanisms of sensing and signalling have been discovered and implicated in a broad range of diseases, from cancer to inflammation and fibrosis, providing opportunities for novel therapeutic avenues. Here, we review some of the most recently discovered mechanisms of lactate sensing and signalling.

Omega-3 polyunsaturated fatty acids reverse the impact of western diets on regulatory T cell responses through averting ceramide-mediated pathways.

Western diet (WD), high in sugar and fat, promotes obesity and associated chronic low-grade pro-inflammatory environment, leading to impaired immune function, reprogramming of innate and adaptive immune cells, and development of chronic degenerative diseases, including cardiovascular disease. Increased concentrations of circulating and tissue ceramides contribute to inflammation and cellular dysfunction common in immune metabolic and cardiometabolic disease. Therefore, ceramide-lowering interventions have been considered as strategies to improve adipose tissue health. Here, we report the ability of omega-3 polyunsaturated fatty acids (n-3PUFA) to attenuate inflammatory phenotypes promoted by WD, through ceramide-dependent pathways. Using an animal model, we show that enrichment of WD diet with n-3PUFA, reduced the expression of ceramide synthase 2 (CerS2), and lowered the concentration of long-chain ceramides (C23-C26) in plasma and adipose tissues. N-3PUFA also increased prevalence of the anti-inflammatory CD4+Foxp3+ and CD4+Foxp3+CD25+ Treg subtypes in lymphoid organs. The CerS inhibitor FTY720 mirrored the effect of n-3PUFA. Treatment of animal and human T cells with ceramide C24 in vitro, reduced CD4+Foxp3+ Treg polarisation and IL-10 production, and increased IL-17, while it decreased Erk and Akt phosphorylation downstream of T cell antigen receptors (TCR). These findings suggest that molecular mechanisms mediating the adverse effect of ceramides on regulatory T lymphocytes, progress through reduced TCR signalling. Our findings suggest that nutritional enrichment of WD with fish oil n-3PUFA can partially mitigate its detrimental effects, potentially improving the low-grade inflammation associated with immune metabolic disease. Compared to pharmacological interventions, n-3PUFA offer a simpler approach that can be accommodated as lifestyle choice.

Differential Metabotypes in Synovial Fibroblasts and Synovial Fluid in Hip Osteoarthritis Patients Support Inflammatory Responses.

Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine-glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine-glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.

Understanding the role of host metabolites in the induction of immune senescence: Future strategies for keeping the ageing population healthy.

Advancing age is accompanied by significant remodelling of the immune system, termed immune senescence, and increased systemic inflammation, termed inflammageing, both of which contribute towards an increased risk of developing chronic diseases in old age. Age-associated alterations in metabolic homeostasis have been linked with changes in a range of physiological functions, but their effects on immune senescence remains poorly understood. In this article, we review the recent literature to formulate hypotheses as to how an age-associated dysfunctional metabolism, driven by an accumulation of key host metabolites (saturated fatty acids, cholesterol, ceramides and lactate) and loss of other metabolites (glutamine, tryptophan and short-chain fatty acids), might play a role in driving immune senescence and inflammageing, ultimately leading to diseases of old age. We also highlight the potential use of metabolic immunotherapeutic strategies targeting these processes in counteracting immune senescence and restoring immune homeostasis in older adults. LINKED ARTICLES: This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Tolerogenic effects of 1,25-dihydroxyvitamin D on dendritic cells involve induction of fatty acid synthesis.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune function, promoting anti-inflammatory, tolerogenic T cell responses by modulating antigen presentation by dendritic cells (DC). Transcriptomic analyses indicate that DC responses to 1,25D involve changes in glycolysis, oxidative phosphorylation, electron transport and the TCA cycle. To determine the functional impact of 1,25D-mediated metabolic remodelling, human monocyte-derived DC were differentiated to immature (+vehicle, iDC), mature (+LPS, mDC), and immature tolerogenic DC (+1,25D, itolDC) and characterised for metabolic function. In contrast to mDC which showed no change in respiration, itolDC showed increased basal and ATP-linked respiration relative to iDC. Tracer metabolite analyses using 13C -labeled glucose showed increased lactate and TCA cycle metabolites. Analysis of lipophilic metabolites of 13C-glucose revealed significant incorporation of label in palmitate and palmitoleate, indicating that 1,25D promotes metabolic fatty acid synthesis in itolDC. Inhibition of fatty acid synthesis in itolDC altered itolDC morphology and suppressed expression of CD14 and IL-10 by these cells. These data indicate that the ability of 1,25D to induce tolerogenic DC involves metabolic remodelling leading to synthesis of fatty acids.

Endothelial cell and T-cell crosstalk: Targeting metabolism as a therapeutic approach in chronic inflammation.

The role of metabolic reprogramming in the coordination of the immune response has gained increasing consideration in recent years. Indeed, it has become clear that changes in the metabolic status of immune cells can alter their functional properties. During inflammation, T cells need to generate sufficient energy and biomolecules to support growth, proliferation, and effector functions. Therefore, T cells need to rearrange their metabolism to meet these demands. A similar metabolic reprogramming has been described in endothelial cells, which have the ability to interact with and modulate the function of immune cells. In this overview, we will discuss recent insights in the complex crosstalk between endothelial cells and T cells as well as their metabolic reprogramming following activation. We highlight key components of this metabolic switch that can lead to the development of new therapeutics against chronic inflammatory disorders. LINKED ARTICLES: This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

Lactate modulation of immune responses in inflammatory versus tumour microenvironments.

The microenvironment in cancerous tissues is immunosuppressive and pro-tumorigenic, whereas the microenvironment of tissues affected by chronic inflammatory disease is pro-inflammatory and anti-resolution. Despite these opposing immunological states, the metabolic states in the tissue microenvironments of cancer and inflammatory diseases are similar: both are hypoxic, show elevated levels of lactate and other metabolic by-products and have low levels of nutrients. In this Review, we describe how the bioavailability of lactate differs in the microenvironments of tumours and inflammatory diseases compared with normal tissues, thus contributing to the establishment of specific immunological states in disease. A clear understanding of the metabolic signature of tumours and inflammatory diseases will enable therapeutic intervention aimed at resetting the bioavailability of metabolites and correcting the dysregulated immunological state, triggering beneficial cytotoxic, inflammatory responses in tumours and immunosuppressive responses in chronic inflammation.

Metabolic Checkpoints in Rheumatoid Arthritis.

Several studies have highlighted the interplay between metabolism, immunity and inflammation. Both tissue resident and infiltrating immune cells play a major role in the inflammatory process of rheumatoid arthritis (RA) via the production of cytokines, adipo-cytokines and metabolic intermediates. These functions are metabolically demanding and require the most efficient use of bioenergetic pathways. The synovial membrane is the primary site of inflammation in RA and exhibits distinctive histological patterns characterized by different metabolism, prognosis and response to treatment. In the RA synovium, the high energy demand by stromal and infiltrating immune cells, causes the accumulation of metabolites, and adipo-cytokines, which carry out signaling functions, as well as activating transcription factors which act as metabolic sensors. These events drive immune and joint-resident cells to acquire pro-inflammatory effector functions which in turn perpetuate chronic inflammation. Whether metabolic changes are a consequence of the disease or one of the causes of RA pathogenesis is still under investigation. This review covers our current knowledge of cell metabolism in RA. Understanding the intricate interactions between metabolic pathways and the inflammatory and immune responses will provide more awareness of the mechanisms underlying RA pathogenesis and will identify novel therapeutic options to treat this disease.

Omega-3 polyunsaturated fatty acids impinge on CD4+ T cell motility and adipose tissue distribution via direct and lipid mediator-dependent effects.

AIMS: Adaptive immunity contributes to the pathogenesis of cardiovascular metabolic disorders (CVMD). The omega-3 polyunsaturated fatty acids (n-3PUFA) are beneficial for cardiovascular health, with potential to improve the dysregulated adaptive immune responses associated with metabolic imbalance. We aimed to explore the mechanisms through which n-3PUFA may alter T cell motility and tissue distribution to promote a less inflammatory environment and improve lymphocyte function in CVMD. METHODS AND RESULTS: Using mass spectrometry lipidomics, cellular, biochemical, and in vivo and ex vivo analyses, we investigated how eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the main n-3PUFA, modify the trafficking patterns of activated CD4+ T cells. In mice subjected to allogeneic immunization, a 3-week n-3PUFA-enriched diet reduced the number of effector memory CD4+ T cells found in adipose tissue, and changed the profiles of eicosanoids, octadecanoids, docosanoids, endocannabinoids, 2-monoacylglycerols, N-acyl ethanolamines, and ceramides, in plasma, lymphoid organs, and fat tissues. These bioactive lipids exhibited differing chemotactic properties when tested in chemotaxis assays with activated CD4+ T cells in vitro. Furthermore, CD4+ T cells treated with EPA and DHA showed a significant reduction in chemokinesis, as assessed by trans-endothelial migration assays, and, when implanted in recipient mice, demonstrated less efficient migration to the inflamed peritoneum. Finally, EPA and DHA treatments reduced the number of polarized CD4+ T cells in vitro, altered the phospholipid composition of membrane microdomains and decreased the activity of small Rho GTPases, Rhoα, and Rac1 instrumental in cytoskeletal dynamics. CONCLUSIONS: Our findings suggest that EPA and DHA affect the motility of CD4+ T cells and modify their ability to reach target tissues by interfering with the cytoskeletal rearrangements required for cell migration. This can explain, at least in part, the anti-inflammatory effects of n-3PUFA supporting their potential use in interventions aiming to address adipocyte low-grade inflammation associated with cardiovascular metabolic disease.

Lactate: Fueling the fire starter.

It is becoming increasingly appreciated that intermediates of metabolic pathways, besides their anabolic and catabolic functions, can act as signaling molecules and influence the outcome of immune responses. Although lactate was previously considered as a waste product of glucose metabolism, accumulating evidence has highlighted its pivotal role in regulating diverse biological processes, including immune cell polarization, differentiation and effector functions. In addition, lactate is a key player in modulating tumor immune surveillance. Hence, targeting lactate-induced signaling pathways is a promising tool to reduce inflammation, to prevent autoimmunity and to restore anti-tumor immune response. This article is characterized under: Biological Mechanisms > Metabolism.

Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring.

Accumulation of lactate in the tissue microenvironment is a feature of both inflammatory disease and cancer. Here, we assess the response of immune cells to lactate in the context of chronic inflammation. We report that lactate accumulation in the inflamed tissue contributes to the upregulation of the lactate transporter SLC5A12 by human CD4+ T cells. SLC5A12-mediated lactate uptake into CD4+ T cells induces a reshaping of their effector phenotype, resulting in increased IL17 production via nuclear PKM2/STAT3 and enhanced fatty acid synthesis. It also leads to CD4+ T cell retention in the inflamed tissue as a consequence of reduced glycolysis and enhanced fatty acid synthesis. Furthermore, antibody-mediated blockade of SLC5A12 ameliorates the disease severity in a murine model of arthritis. Finally, we propose that lactate/SLC5A12-induced metabolic reprogramming is a distinctive feature of lymphoid synovitis in rheumatoid arthritis patients and a potential therapeutic target in chronic inflammatory disorders.

Fatty acids - from energy substrates to key regulators of cell survival, proliferation and effector function.

Recent advances in immunology and cancer research show that fatty acids, their metabolism and their sensing have a crucial role in the biology of many different cell types. Indeed, they are able to affect cellular behaviour with great implications for pathophysiology. Both the catabolic and anabolic pathways of fatty acids present us with a number of enzymes, receptors and agonists/antagonists that are potential therapeutic targets, some of which have already been successfully pursued. Fatty acids can affect the differentiation of immune cells, particularly T cells, as well as their activation and function, with important consequences for the balance between anti- and pro-inflammatory signals in immune diseases, such as rheumatoid arthritis, psoriasis, diabetes, obesity and cardiovascular conditions. In the context of cancer biology, fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of these highly proliferating cells. Fatty acids can also be involved in a broader transcriptional programme as they trigger signals necessary for tumorigenesis and can confer to cancer cells the ability to migrate and generate distant metastasis. For these reasons, the study of fatty acids represents a new research direction that can generate detailed insight and provide novel tools for the understanding of immune and cancer cell biology, and, more importantly, support the development of novel, efficient and fine-tuned clinical interventions. Here, we review the recent literature focusing on the involvement of fatty acids in the biology of immune cells, with emphasis on T cells, and cancer cells, from sensing and binding, to metabolism and downstream effects in cell signalling.

Selective neuronal silencing using synthetic botulinum molecules alleviates chronic pain in mice.

Chronic pain is a widespread debilitating condition affecting millions of people worldwide. Although several pharmacological treatments for relieving chronic pain have been developed, they require frequent chronic administration and are often associated with severe adverse events, including overdose and addiction. Persistent increased sensitization of neuronal subpopulations of the peripheral and central nervous system has been recognized as a central mechanism mediating chronic pain, suggesting that inhibition of specific neuronal subpopulations might produce antinociceptive effects. We leveraged the neurotoxic properties of the botulinum toxin to specifically silence key pain-processing neurons in the spinal cords of mice. We show that a single intrathecal injection of botulinum toxin conjugates produced long-lasting pain relief in mouse models of inflammatory and neuropathic pain without toxic side effects. Our results suggest that this strategy might be a safe and effective approach for relieving chronic pain while avoiding the adverse events associated with repeated chronic drug administration.

Neuroprotective Properties of a Macrolide Antibiotic in a Mouse Model of Middle Cerebral Artery Occlusion: Characterization of the Immunomodulatory Effects and Validation of the Efficacy of Intravenous Administration.

Repurposing the macrolide antibiotic azithromycin has recently been suggested as a promising neuroprotective strategy for the acute treatment of ischemic stroke. Here, we aim at further characterizing the immunomodulatory properties of intraperitoneal (i.p.) administration of this drug and, more importantly, at assessing whether neuroprotection can also be achieved by the more clinically relevant intravenous (i.v.) route of administration in a mouse model of focal cerebral ischemia induced by transient (30-min) middle cerebral artery occlusion (MCAo). A single i.p. injection of azithromycin (150 mg/kg) upon reperfusion prevented ischemia-induced spleen contraction and increased the number of MAC-1-immunopositive microglia/macrophages in the ischemic hemisphere 48 h after the insult. This was paralleled by an elevation of alternatively activated phenotypes (i.e., Ym1-immunopositive M2-polarized cells) and by a reduced expression of the pro-inflammatory marker myeloperoxidase. More importantly, i.v. administration of azithromycin upon reperfusion reduced MCAo-induced infarct volume and cerebral edema to an extent comparable to that obtained via the i.p. route. Although the i.p. route is often used for research purposes, it is impractical in the clinical setting; however, i.v. administration can easily be used in ischemic stroke patients who usually have i.v. access already established on hospital admission. The neuroprotective efficacy of the clinically relevant i.v. administration of azithromycin, together with its beneficial immunomodulatory properties reported in mice subjected to transient MCAo, suggests that this macrolide antibiotic can be effectively repurposed for the acute treatment of ischemic stroke. To this end, further work is needed to validate the efficacy of azithromycin in the clinical setting.

Caspase-1-independent Maturation of IL-1ß in Ischemic Brain Injury: is there a Role for Gelatinases?

Ischemic stroke is a devastating condition primarily caused by reduced blood supply to the brain. Interleukin (IL)-1ß is a pro-inflammatory cytokine that plays a pivotal role in the detrimental inflammatory processes that participate to cerebral ischemic damage. After injury, it is produced by distinct cells of the neurovascular unit as an inactive precursor, pro-IL-1ß. Although previous studies have suggested that caspase-1 is the main enzyme implicated in the cleavage of pro-IL-1ß into the biologically active cytokine, recent work has demonstrated that, under ischemia-reperfusion conditions, other mechanisms may be involved in cytokine maturation. Indeed, we have shown that in rats subjected to transient middle cerebral artery occlusion (MCAo), elevation of IL-1ß levels is paralleled by an elevation of gelatinolytic, but not caspase-1 activity in the injured hemisphere and pharmacological inhibition of gelatinases, i.e. matrix metalloproteases (MMP)-2 and MMP-9 prevents cytokine maturation. These findings further support the hypothesis that, under ischemia-reperfusion injury, cerebral elevation of IL-1ß occurs via mechanisms other than caspase-1, likely involving gelatinases.

Azithromycin protects mice against ischemic stroke injury by promoting macrophage transition towards M2 phenotype.

To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotective effects of the macrolide antibiotic azithromycin in a mouse model system of transient middle cerebral artery occlusion. Intraperitoneal administration of azithromycin significantly reduced blood-brain barrier damage and cerebral infiltration of myeloid cells, including neutrophils and inflammatory macrophages. These effects resulted in a dose-dependent reduction of cerebral ischemic damage, and in a remarkable amelioration of neurological deficits up to 7 days after the insult. Neuroprotection was associated with increased arginase activity in peritoneal exudate cells, which was followed by the detection of Ym1- and arginase I-immunopositive M2 macrophages in the ischemic area at 24-48 h of reperfusion. Pharmacological inhibition of peritoneal arginase activity counteracted azithromycin-induced neuroprotection, pointing to a major role for drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype.

Activation of RXR/PPARγ underlies neuroprotection by bexarotene in ischemic stroke.

The identification of novel drug targets for the treatment of ischemic stroke is currently an urgent challenge. Recent experimental findings have highlighted the neuroprotective potential of immunomodulatory strategies, based on polarization of myeloid cells toward non-inflammatory, beneficial phenotypes. Given the role of retinoid X receptors (RXR) in myeloid cells differentiation and polarization, here we have explored the neuroprotective potential of the RXR agonist bexarotene in mice subjected to focal cerebral ischemia. Acute administration of bexarotene significantly reduced blood brain barrier leakage, brain infarct damage and neurological deficit produced by transient middle cerebral artery occlusion in mice, without affecting cerebral blood flow. The rexinoid exerted neuroprotection with a wide time-window, being effective when administered up to 4.5h after the insult. The amelioration of histological outcome, as well as the ability of bexarotene to revert middle cerebral artery occlusion (MCAo)-induced spleen atrophy, was antagonised by BR1211, a pan-RXR antagonist, or by the selective peroxisome proliferator-activated receptor (PPAR)γ antagonist bisphenol A diglycidyl ether (BADGE), highlighting the involvement of the RXR/PPARγ heterodimer in the beneficial effects exerted by the drug. Immunofluorescence analysis revealed that bexarotene elevates Ym1-immunopositive N2 neutrophils both in the ipsilateral hemisphere and in the spleen of mice subjected to transient middle cerebral artery occlusion, pointing to a major role for peripheral neutrophil polarization in neuroprotection. Thus, our findings suggest that the RXR agonist bexarotene exerts peripheral immunomodulatory effects under ischemic conditions to be effectively repurposed for the acute therapy of ischemic stroke.